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From 2010.igem.org

5/7 Membership meeting New participant of CBNU-KOREA team. Introduced what is the iGEM, what we should do if we join the competition. 5/15 Concept meeting Decided 2010 CBNU-KOREA iGEM team’s project(almost 4hr) 5/18 Researching Finding related articles, paper and so on. Essential gene data search 5/25 Strategy about database Planned how to re-construct essential gene database. 7/19 Plasmid Culture • Pick up a single colony from fresh cultured LB agar plate. • Our established media and inoculate the cell into the 5ml of fresh LB liquid media (contains antibiotics) and our established media at 37℃ with shaking for 12hr. • plasmid : pSB1C3, pSB1A3, pSB1T3, pSB1K3 (contain J54450) 7/20 Plasmid extract • Uses kit and extracted plasmid. (pSB1C3, pSB1A3, pSB1T3, pSB1K3) • Confirmed size by electrophoresis. 7/23 Plasmid Digest • Digest of 4 plasmid. • Used EcoRⅠ-PstⅠ restriction endonuclease. • Confirmed size by electrophoresis. 7/24 Primer Design • Designed Vibrio cholerae's oriC2vc, parA, parB, parS, dif gene primer using ‘Ginko primer’. 7/28 DNA Amplification • Amplified ori, parA, parB, parS, dif gene of vibrio cholerae. • Confirmed size by electrophoresis. Concentration • Because concentration of parS, dif gene was low, it was hard to confirm result by electrophoresis. • Concentrated gene sample 7/29 DNA(gene) Digest • Digest of 4 plasmid. • Used EcoRⅠ-PstⅠ restriction endonuclease. • Confirmed size by electrophoresis. 7/30 Prepared Compentent cell • E.coli DH10B, DH5α, JM109 8/2 DNA Ligation/Transformation • Ligation inserting gene in vector (parA+pSB1A3, parS+pSB1C3, ori+pSB1K3, dif+pSB1A3) • Transformation (Competent cell : E.coli DH10B) • Spreading on plate (each antibiotics contained LB agar media) 8/3 Cell Culture • Pick up a single colony from fresh cultured LB agar plate. • Our established media and inoculate the cell into the 5ml of fresh LB liquid media (contains antibiotics) and our established media at 37℃ with shaking for 12hr. • vector : parA+pSB1A3, parS+pSB1C3, ori+pSB1K3, dif+pSB1A3 8/4 Vector extract • Uses kit and extracted plasmid. (parA+pSB1A3, parS+pSB1C3, ori+pSB1K3, dif+pSB1A3) • Confirmed size by electrophoresis. 8/9 Plasmid Digest • Digest of 4 vector. • ori+pSB1K3, parA+pSB1A3, parS+pSB1C3 digests by EcoRⅠ-SpeⅠ restriction endonuclease and dif+pSB1A3 gene digested by XbaⅠ-PstⅠ restriction endonuclease. • Confirmed size by electrophoresis. • Came out as size of ori+pSB1K3 gene is smaller. 8/10 Sequencing Order • Analyzed plasmid DNA extraction (cultured cell) 8/16 Primer Again design • According to analysis result, primer of ori gene knew wrong fact. • Designed primer of ori gene again. • Primer of I51020 and p1003 designed. 8/20 DNA Amplification • Amplified I51020, p1003 • Confirmed size by electrophoresis. 8/23 Plasmid Digest • I51020 digests by EcoRⅠ-PstⅠ restriction endonuclease. • p1003 digests by XbaⅠ-PstⅠ restriction endonuclease. • Confirmed size by electrophoresis. 8/24 DNA Ligation/Transformation • Ligated inserting gene in vector (Inserted ori gene and p1003 antibiotic resistance cassette in I51020 vector, inserted parS gene and dif gene in pSB1A3 vector) • Transformation (Competent cell : E.coli DH10B) • Spreading on plate (each antibiotics contained LB agar media)